Enhancing anti-AML activity of venetoclax by isoflavone ME-344 through suppression of OXPHOS and/or purine biosynthesis

Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these combination therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 synergized with VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, an inhibitor of purine biosynthesis, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired resistance to AraC showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. These results translated into significantly prolonged survival upon combination of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.


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The 5-year overall survival (OS) rate for acute myeloid leukemia (AML) remains low for adults 60 (<30%) and worse for elderly patients (<10%). 1,2 For AML patients who are ≥75 years of age or  The second-generation isoflavone, ME-344, has been reported to inhibit oxidative 69 phosphorylation (OXPHOS) in solid tumors 8,9 and was tested in Phase I clinical trials 70 (NCT01544322, NCT02806817, NCT02100007). It showed significant antitumor activity after 71 bevacizumab treatment in patients with early HER2-negative breast cancer. 10 In a basket trial of 72 advanced/metastatic small cell lung, ovarian and cervical cancers, ME-344 was well tolerated with 73 11 out of 21 evaluable patients having either a partial response (1 patient) or stable disease (10 74 patients) after 3 cycles of treatment. 11 In AML, a preclinical study reported that ME-344 was 75 cytotoxic to cell lines and primary patient samples while sparing normal hematopoietic cells. 12 76 However, it remains unknown if ME-344 also reduces OXPHOS in AML cells. 12

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Since recent studies suggest that targeting OXPHOS enhances the antileukemic activity of 78 VEN, 13-15 we aimed in this study to investigate the antileukemic activity of combined ME-344 and 79 VEN against AML cells and the underlying molecular mechanisms.   105 MTT (3-[4,5-dimethylthiazol-2-yl]−2,5-diphenyltetrazolium bromide; Sigma-Aldrich) assays 106 were performed as described. 19,22 Primary AML cells were treated with ME-344, VEN, ME-107 344+VEN or vehicle for 72 hours, followed by MTT analysis. The extent and direction of the 108 antileukemic interactions were determined by isobologram analyses, as described. 22 were extracted using 80% methanol, as described. 19 Primary cells, AML and normal human bone marrow cells (BM), were seeded in 24-well 127 plates in triplicate at the density of 300,000-500,000 cells/mL and treated with ME-344 or DMSO 128 (<0.5% (v/v)) for 8 hours, followed by VEN addition, for a total of 48 hours. Cells were then 129 washed twice with Annexin V Binding Buffer (AVBB), stained with 100 µL of the antibody mix:  133 AML cell lines were pre-treated with ME-344 for 8 hours, followed by VEN treatment (1 hour).

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The cells were then stained with 10 µg/mL of JC-1 dye (Thermo Fisher Scientific) in PBS (37˚C, 135 20 minutes), washed, and analyzed by flow cytometry.   were suspended at the onset of leukemia symptoms (hindleg weakness, >5-10% weight loss, 164 ruffled fur). Mice were euthanized when leukemic symptoms progressed (hindleg paralysis, >10-165 15% weight loss, internal mass >500 mg); with all outcomes confirmed by necropsy. All mice 166 were provided food and water ad libitum, given supportive fluids and supplements as needed, and

Statistical analyses
173 Unpaired t-tests were used for comparisons between two treatment groups. One-way ANOVA 174 with Bonferroni correction was used for comparisons between four treatment groups. Error bars 175 represent mean ± standard error; the significance level was set at p<0.05. Overall survival was 176 estimated using the Kaplan-Meier method, and statistical analysis was performed using the log-177 rank test. Precent increase in lifespan was determined using the formula: treated mediancontrol 178 median/control median x 100; median = median day of death due to leukemic progression. All

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To determine whether ME-344 enhances the antileukemic activity of VEN, AML cell lines were 189 treated for 24 hours with ME-344 and VEN, alone or combined, and subjected to apoptosis primary naïve AML patient samples treated with ME-344, VEN, or ME-344+VEN for 72 hours.

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Standard isobologram analyses revealed synergy between ME-344 and VEN in both primary AML 197 patient samples ( Figure 1C).

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To further support the clinical relevance and estimate therapeutic window, normal human   Figure 2D).

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To determine the mechanism of action of the ME-344 and VEN combination, the XFe96 showed similar significant decrease of ΔΨm ( Figure 3F). In primary AML patient sample 250 AML#16, ME-344 significantly suppressed basal OCR and SRC, along with a borderline 251 significant suppression of maximal OCR (p=0.065) but showed no effect on OCR related to ATP 252 production ( Figure 3G-H). Further, its combination with VEN did not cause further suppression 253 of OCR. In primary AML patient sample AML#17, although ME-344 did not show any impact on 254 OCR, it significantly enhanced the suppression of maximal OCR and SRC by VEN ( Figure 3I-J).

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To determine the importance of OXPHOS suppression for ME-344 and VEN-induced 256 apoptosis, we forced cells to rely on OXPHOS by substituting glucose in culture media with 257 galactose. 34-36 MV4-11 cells incubated in media supplemented with either glucose or galactose 258 were pretreated with ME-344 (8 hours), followed by the addition of VEN (16 hours; Figure S2A).  Table S2).  Figure 4F).

ME-344 enhances the antileukemic activity of VEN against AraC-resistant AML cells 284
Resistance to chemotherapy drugs remains a major cause of treatment failure in AML. Therefore, 285 identifying therapies that can target chemotherapy-resistant AML is critical for the improvement  To determine whether ME-344 targets AraC-resistant AML cells and enhances VEN 301 activity, parental and AraC-resistant cells were treated with ME-344 (24 hours). ME-344-induced 302 apoptosis was significantly higher in AraC-resistant cells than the parental cells ( Figure 5D). dose schedule for ME-344. Treatment was initiated on day 4 via either ip or iv administration.

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Doses were escalated based on mouse body weights and development of symptoms ( Figure S3A).

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Of note, mice in the high dose ip arm had three deaths (day 25, 25, and 28; necropsy determined 324 deaths were due to drug toxicity; LD60), while the lower dose prolonged median survival (MS) = 325 36 days, mice needed longer recovery time ( Figures S3B-C). The iv arm also prolonged survival 326 (37 days) compared to control (31 days; Figure S3D) with no adverse symptoms, so ME-344 at 327 200 mg/kg (iv) ME-344 was selected as the dose for further studies.

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To evaluate the efficacy of ME-344+VEN combination in vivo, mice engrafted with MV4-           The combination of ME-344 and VEN induces apoptosis in a time-dependent fashion. (A) MV4-11 and THP-1 cells were simultaneously treated with ME-344 and VEN for various times, ranging from 1-12 hours. Treated cells were stained with Annexin V-FITC/PI and subjected to ow cytometry analyses. # indicates p<0.05, ## indicates p<0.01, and ### indicates p<0.001 compared to single drug treatments of the same treatment time. * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.001 compared to vehicle control treated cells of the same treatment time. ns indicates not signi cant. (B) Sequential treatment regimen described for experiments in panel C. (C) Annexin V/PI staining and ow cytometry analyses of cells pretreated with ME-344 for 8 hours followed by the addition of VEN for 1 or 2 hours. # indicates p<0.05, ## indicates p<0.01, and ### indicates p<0.001 compared to single drug treatments of the same treatment time. * indicates p<0.05 and *** indicates p<0.001 compared to vehicle control treated cells. (D) Annexin V/PI staining and ow cytometry analyses of cells pretreated with ME-344 for 8 hours followed by the addition of VEN for 16 hours, totaling 24 hours of treatment. ** indicates p<0.01 and *** indicates p<0.001 compared to vehicle control and single drug treated cells. Flow experiments shown in panels A, C, and D were performed three times in triplicate; data from one representative experiment is shown.